GSM7049567: 017_tumor; Mus musculus; OTHER
Two months old Glut1F/F mice (control mice) and Glut1F/FPRβ-CreERT2 mice (conditional Glut1 knockout mice) were received implantation of MC38 murine colorectal cancer cells by portal vein injection. Cryo-sections of MC38 liver metastases were fixed with formalin, hybridized with NanoString mouse whole transcriptome atlas, followed by immunofluorescence staining with anti-desmin to label CAFs, anti-CD45 for immune cells, and SYTO 13 for cell nuclei. Regions of Interest (ROIs) were selected for data requisition and analysis. CAFs (Desmin+ CD45-) within a ROI were collected for transcriptomic profiling and MC38 cancer cells (Desmin- CD45-) within a same ROI were collected for transcriptomic profiling as well. Segments 17-20 were ROIs from Glut1F/FPRβ-CreERT2 mice and 21-26 were from Glut1 F/F mice. 20 sequencing data were generated for the CAFs due to dual-indexing primers-based sequencing, which was done on Illumina NextSeq 2000. immunofluorescence (IF) staining was performed using commercially available antibodies against Desmin (Ab32362 abcam, 1:400 dilution) and CD45 (NovusBio, NBP1-44763).  Slides were blocked with Buffer W probes (NanoString Technologies, Cat#121300313) at room temperature for 2 hours.  Anti-desmin unconjugated primary antibody was incubated overnight at 4°C.  Slides were washed three times in 2XSSC and then incubated for 2 hours with anti-rabbit secondary antibody conjugated to Alexa Fluor 647 (Thermofisher, # A-21244) (1:500 dilution), CD45 directly conjugated antibody (AF532) and SYTO 13 fluorescent nucleic acid stain (ThermoFisher, Cat# S7575) were followed Prepared tissues were flooded with whole transcriptome RNA probes (NanoString Technologies, Cat#121401102) attached to oligonucleotide barcodes via photocleavable linkers.  Slides were covered with HybriSlip hybridization covers (Grace Bio-Labs, Cat#714022) and hybridized overnight at 37°C in a hybridization oven (Boekel Scientific RapidFISH Slide Hybridizer, Cat#240200).  Following hybridization, unbound probes were washed away using a solution of equal parts 4XSSC and deionized formamide (ThermoFisher, Cat#AM9342) Slides were loaded and scanned in the Nanostring GeoMx DSP instrument.  Exposure times for each channel were optimized and set to 100 ms (FITC), 600 ms (Texas Red) and 250 ms (Cy5).  Circular ROIs ranging form approximately 400 – 660 microns were drawn on the resulting images utilizing Desmin IF staining for guidance.  ROI masks were created using the GeoMx DSP Control Center software by utilizing the fluorescence intensity of the immunofluorescence staining.  The thresholding of each segment was adjusted for each ROI to accommodate for IF staining heterogeneity. Segments were selected for CAFs (desmin positive and CD45 negative regions), and tumor regions (desmin and CD45 negative regions; reminder).  Selected segments were illuminated by UV laser in the GeoMx instrument and photocleaved barcodes were collected and deposited into a 96-well microplate by the instruments onboard microfluidics system.  Collection plates were sealed to prevent contamination and stored at -80°C prior to library preparation.